DESCRIPTION: Any therapeutic regimens against HIV must address the issue of viral resistance and escape. Gene therapy is an attractive alternative or adjunct to combination drug therapy because large number of antiviral genes can be devised that target viral as well as host cell sequences. We have been developing optimized ribozymes (Rz's) that recognize the HIV genome at multiple conserved sites, and more recently, Rz's that cleave the chemokine receptor, CCR5, which is a critical co-receptor for primary HIV infection. Individually, these genes have been shown to effectively inhibit HIV replication in T cells, and ongoing efforts involve assembly of several of these genes into a single retroviral vector for clinical evaluation. Here we propose to construct and validate the safety and efficacy of a polyribozyme vector for eventual clinical application. In parallel, we will design the next generation of polyRz vectors, which may include ones designed against possible resistant virus mutations and additional co-receptors. The specific aims of project 1 are: (1) To construct and test a murine retroviral vector expressing polyribozymes (4 anti-HIV and 1 anti-CCR5) for efficacy and safety at the preclinical level. (2) To identify resistant mutants to the U5, vif and pol Rz's by randomization of the target sequences in an infectious HIV-1 clone and passage in Rz expressing cells. If mutations are identified, ribozymes targeting them will be designed and tested. (3) To examine the effects of constitutive and inducible expression of ribozymes against CCR5, CCR3 and CXCR-4 on helper T cell functions of antigen-specific CD4 cell clones and on hematopoietic progenitor cells on their proliferation, differentiation and progeny cell function in vitro. Thus, our goals are to prepare and validate the first generation of polyRz vector as well as to design the next generation of polyRz vector which incorporate Rz's against potential resistant mutations and other co-receptors.